Figure 2.

EZH2 depletion abolished p21 and Chk1 activation in response to DNA damage. (a) Western blot analysis showing that EZH2 depletion abolished ADR (1 μM)- or ETO (10 μM)-induced p21 accumulation and Chk1 phosphorylation, and increased levels of phosphorylated H2AX and PARP cleavage. β-Actin is shown as loading control. (b) Western blot analysis in Saos-2 cells showing that EZH2 depletion abrogated ADR-induced Chk1 phosphorylation, and increased levels of phosphorylated H2AX and PARP cleavage. β-Actin is shown as loading control. (c) Western blot analysis showing the effects of another EZH2 siRNA (#2) targeting 5′-untranslated region of EZH2 mRNA on cell cycle checkpoint proteins. (d) Western blot analysis showing the rescue effects of EZH2 and EZH2 SET domain deletion (EZH2Δ) on p21 and Chk1. FACS analysis in U2OS showing the effects of EZH2 depletion on checkpoint abrogation (e) and apoptosis (f) are rescued by wild-type EZH2, but no by EZH2Δ. Data are means±S.D. (n=3), ^*P<0.05 (Student's t-test). (g) Quantitative real-time PCR (qRT-PCR) analysis of p21 mRNA levels in NC- or siEZH2-treated U2OS cells in the presence or absence of ADR (1 μM) treatment for 24 h. Data are means±S.D. (n=3). (h) Western blot analysis of p21 and Chk1 in U2OS cells treated with ADR and EZH2 siRNA in the presence or absence of proteasome inhibitors MG132 (5 μM) or MG115 (10 μM)